Fluid stream imaging apparatus

ABSTRACT

A fluid stream imaging apparatus having either optics for manipulating the aspect ratio or sensing elements configured for manipulating the aspect ratio of an image of the fluid stream. This application may also relate to a system for acquiring images of a portion of a fluid stream at high speeds for image processing to measure and predict droplet delays for individual forming particles.

This application is a continuation of and claims the benefit of priorityto U.S. Ser. No. 13/366,124, filed on Feb. 3, 2012 which claims thebenefit of priority to U.S. Provisional Patent Application Ser. No.61/439,757, filed on Feb. 4, 2011, the contents of each application areherein incorporated by reference in their entirety.

FIELD

The present disclosure generally relates to the field of flow cytometry,and more particularly a fluid stream imaging apparatus.

BACKGROUND

Various flow cytometers and microfluidic systems exist for the purposeof analyzing and sorting particles. Each of these instruments hasvarious shortcomings relating to their ability to maintain accurate sortactions. Jet-in-air flow cytometers are commonly used for the purpose ofsorting particles based on detected characteristics. The operation of ajet-in-air flow cytometer may involve producing a coaxial fluid streamin a nozzle. The coaxial fluid stream has a core stream of sample, whichincludes the particles or interest, and an outer stream of sheath fluid.

The sheath fluid provides a means for positioning particles andpreventing clogging in the nozzle, as well as, for providing conductivemedium suitable for retaining an applied charge.

The coaxial fluid stream may be perturbed with an oscillator, such as apiezoelectric crystal, resulting in the formation of droplets downstreamof a nozzle. The droplets may contain individual particles or a smallgroup of particles. Based on a desired sort action, each droplet may becharged just prior to separating from the fluid stream at a break offpoint. The appropriate time for applying this charge is known as thedrop delay. As droplets may be formed at a rate of between about 20,000per second and 200,000 per second, the drop delay must be very preciselycalculated.

Historically, the drop delay was determined through an iterative seriesof protocols largely consisting of trial and error test streams havingvarying drop delays. Beads or particles were run through test streamsand collected in puddles. The number of beads or particles collected ineach puddle provided an indication of the drop delay. Such manualprotocols are time intensive and may be lacking in the precisionrequired to achieve extremely accurate sort decisions and are incapableof real time verifications or adjustments.

U.S. Pat. No. 6,248,590 describes an attempt to monitor the drop delaywith the use of a single camera for imaging a portion of the fluidstream, or with multiple cameras for imaging separate portions of thefluid stream, such as for determining the speed of particles at thenozzle and at the speed of particles at a drop off point. From thisinformation an approximation is derived utilizing an exponential decaymodel. However, the formation of droplets may not be so easilypredictable and because only a portion of the stream is monitored,upstream changes may not be detected reliably.

U.S. Patent application publication 2001/02218892 provides a cameramounted on a movable stage for taking multiple images of a fluid stream.The images are then stitched together and the widths are determined.From this information a number of droplets, peaks, and periods betweenthe inspection zone and the drop delay maybe determined. Thisconfiguration provides for means of producing a composite image of theentire fluid stream, but is lacking the ability to monitor the fluidstream in real time because the camera must traverse the fluid stream ata rate thousands of times slower than the fluid stream itself. Further,a stitched together image may not accurately reflect the stream at anyparticular time and is time consuming to produce. Changes in operatingconditions, such as oscillator frequency, oscillator amplitude,temperature, surface tension, and harmonic conditions may vary thestream during the time in which the individual images are beingcaptured. Positional changes in the excitation source or the dropletbreak off point during operation, upon start up, or during other changescannot quickly be realized by the described system because a series ofimages must first be captured and then stitched together.

SUMMARY OF INVENTION

Certain aspects of this disclosure relate to an improved system forimaging a fluid stream, such as in a flow cytometer for analyzing and/orsorting particles. The provisions of such a system, in one embodimentinclude: a fluid delivery system having a nozzle for producing a fluidstream, the fluid delivery system having an oscillator for perturbingthe fluid stream into droplets at a break off point downstream of thenozzle; an excitation energy source for interrogating the fluid streamat an inspection zone; an optical system for manipulating the aspectratio of an image of the fluid stream; and a sensing element for sensingthe manipulated image of the fluid stream. Such a system may be capableof producing an image of a fluid stream with sufficient detail along theflow axis of the stream and across the flow axis of the stream forderiving an accurate break off point and an accurate drop delay.

Other aspects of this disclosure relate to an improved imaging devicefor a fluid stream, such as in a flow cytometer for analyzing andsorting particles. The provisions of such a system, in one embodimentinclude: a fluid delivery system having a nozzle for producing a fluidstream, the fluid delivery system having an oscillator for perturbingthe fluid stream into droplet at a break off point downstream of thenozzle; an excitation energy source for interrogating the fluid streamat an inspection zone; an optical system for producing a image of thefluid stream, the image of the fluid stream including at least theinspection zone and the break off point; and a high resolution sensingelement for sensing an image of the fluid stream.

Still other aspects of this disclosure may relate to a system ofprocessing digital images in real time to predict the drop delay of eachforming droplet. Such a system may include: a fluid delivery systemhaving a nozzle for producing a fluid stream, the fluid delivery systemhaving an oscillator for perturbing the fluid stream into droplet at abreak off point downstream of the nozzle; an excitation energy sourcefor interrogating the fluid stream at an inspection zone; a detector fordetecting electromagnetic radiation from particles at the inspectionzone and for determining particle characteristics; a controller forcharging the fluid stream based on the detected particlecharacteristics; an optical system for producing a image of the fluidstream, the image of the fluid stream including at least the break offpoint, wherein the optical system takes multiple images per droplet; asensing element for sensing a plurality of images of the fluid stream;and a processor for processing the detected image of the fluid stream,wherein the processor includes is responsive to images of the fluidstream to determine and/or predict the time at which a droplet breaksoff the fluid stream.

One broad object provided in this disclosure can be a camera to producea single image of a fluid stream, where that image includes both aninterrogation point, or inspection zone, and the break off point, andwhere the necks of forming droplets are captured with sufficientresolution across the flow axis (stream width) for precisely determiningor updating a drop delay. From this image changes in the distance fromthe inspection zone to the break off point may provide more accuratedrop delay information than previous systems.

An object of this disclosure can be to provide an apparatus and methodfor performing more accurate sort actions by precisely monitoring thefluid stream. Specifically, one broad object of the apparatus providedherein is to provide the capability for manipulating an image of theentire fluid stream into a format allowing important features throughoutthe entire fluid stream to be monitored in real time.

Another broad object provided herein may be to image the entire fluidstream, or part of the fluid stream, and to provide sufficientresolution to monitor droplets forming in real time, so that a specificdrop delay can be determined, or predicted, for each sorted droplet.Images may be processed digitally to determine changes in the drop delayor other parameters such as the location of the break off point orlocation of the inspection zone.

A broad object of this disclosure can be to provide an apparatus forsorting particles and a method of sorting particles which meets theneeds described above. Naturally, further objects of the disclosure areprovided throughout the specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a flow cytometer with a fluid stream imagingapparatus in accordance with certain embodiments described herein.

FIG. 2 illustrates an embodiment of a fluid stream imaging apparatus inaccordance with certain embodiments described herein.

FIG. 3 illustrates an embodiment of a fluid stream imaging apparatus inaccordance with certain embodiments described herein.

FIG. 4 illustrates a manipulated image of a fluid stream with acontinuous change in the aspect ratio along the flow axis.

FIG. 5 illustrates a manipulated image of a fluid stream with a singledisjointed change in the aspect ratio along the flow axis.

FIG. 6 illustrates an embodiment of a fluid stream imaging apparatus inaccordance with certain embodiments described herein.

FIG. 7 illustrates an embodiment of a fluid stream imaging apparatus inaccordance with certain embodiments described herein.

FIG. 8 illustrates a flow chart of a method in accordance with certainembodiments described herein.

MODES FOR CARRYING OUT THE INVENTION

The embodiments described herein relate to the analysis and sorting ofparticles, such as by flow cytometry. A number of inventive conceptsprovided below may be combined or applied to sorting systems other thanflow cytometers.

Now referring primarily to FIG. 1, an example of a flow cytometer 10 isillustrated as a jet-in-air flow cytometer. The flow cytometer 10 mayinclude a fluid delivery system 12, such as a nozzle 16 having a nozzleorifice 24 for delivering a fluid stream 18 to a detection system 14.The fluid stream 18 may be perturbed into droplets 28 by an oscillator26. The droplets 28 may pass through an electromagnetic field producedby deflection plates 40. A charge applied to each droplet 28 will definea path into one of one or more collection containers 42.

The fluid stream 18 may comprise a coaxial fluid steam having an innercore stream of sample 20 and an outer stream of sheath fluid 22. Thefluid stream 18 may exit the nozzle orifice 24 with increasinglypronounced undulations 104 or decreasing neck 106 thicknesses in adownstream direction until a break off point 30 is reached wheredroplets 28 break away from the fluid stream 18. The break off point 30is illustrated as the last point at which a droplet 28 contacts thefluid stream 18. In the field of flow cytometry, this locationrepresents the last point in time a charge may be applied to a droplet28.

The detection system 14 may include an excitation energy source 32 forproviding energy, such as a laser, a light emitting diode, or an arclamp, to the fluid stream 18 and particles of interest contained in thesample 20. The excitation energy source 32 is aligned with an inspectionzone 34 on the fluid stream 18 for interrogating particles as they passthe inspection zone 34. The inspection zone 34 may be located downstreamof the nozzle orifice 24 or may be located within a cuvette or a flowchamber upstream of the nozzle orifice 24. Reflected or emittedelectromagnetic radiations from the fluid stream 18 and particles in thefluid stream 18 can be collected by a detector 36. The detector 36 mayinclude any number of detectors configured in the forward, side, or backdirection relative to the excitation energy source 32. Various optics,such as filters, minors, dichoric mirrors, splitters, and otherreflective and refractive elements, may be used to detectelectromagnetic radiation at any number of wavelengths and/or in anynumber of directions and in a variety of combinations.

Detected signals may be processed for the classification of particleswithin the fluid stream 18 and sort decisions may be made at acontroller 38. The controller 38 may include acquisition and sortelectronics in the form of analog or digital components for processingsignals from the detector and applying a sort logic. Once a sortdecision is made, the controller 38, may act to charge the fluid stream18 through the sample 22 at in the nozzle 16 so that the droplets 28 aredeflected by deflection plates 40 into the appropriate container 42.

The timing at which the appropriate charge is applied to the fluidstream 18 must be closely matched to the time a particle is in a dropletat the break off point 30 in order to ensure an accurate sort action. Animaging device 102 may be provided to monitor or update the distancebetween the break off point 30 and the inspection zone 34 and the numberof undulations 104 in the fluid stream 18 to predict a current orupdated drop delay. The imaging device 102 may include an optical system44 and a sensing element 48 for capturing an image of the fluid stream46 for the purpose of modifying or detecting the appropriate drop delayfor accurate sort decisions.

A strobe 80 may illuminate the fluid stream 18 at predicted intervals tocreate an image of the fluid stream 46 as photons reflected from theobject of the fluid stream 18. With reference to the figures, the fluidstream, otherwise known as the object, is indicated as characterreference 18, while an image of the fluid stream within a field of viewis indicated as character reference 46. When character references 18 and46 are directed to the same portion of a figure, character reference 18should be understood to represent the object of the fluid stream, whilecharacter references 46 should be understood to represent the image ofthe fluid stream as it would be captured or sampled by the optic system44. The optical system 44 may include a series of optical elements formanipulating the image of the fluid stream 46. As one example, theoptical system 44 may comprise multiple lenses or multiple mirrors,other reflective or refractive elements, and combinations of differentreflective and refractive elements. In one embodiment, the opticalsystem 44 may manipulate the aspect ratio of the image of the fluidstream 46, such as compressing the length 68 (seen in FIG. 2) of thefluid stream and expanding the width 70 (seen in FIG. 2) of the fluidstream. By manipulating the aspect ratio to form a manipulated image ofthe fluid stream 50, the optical system 44 may serve to preserverelevant information pertaining to the drop delay. As one non-limitingexample, the length 68 (along the flow axis) of the image of the fluidstream 46 may be compressed by a factor of about 2 times (or magnifiedby a factor of 0.5). Such a compression may allow for the entire lengthof the fluid stream 18 to be captured in an image with sufficientresolution to identify and digitally process undulations 104 and necks106 in the fluid stream 18. At the same time, the width 70 (across theflow axis) may be magnified by a factor of 5 in order to amplifyfeatures such as the thickness of the necks 106 in the fluid stream 18.Such an optical system 44 for modifying an image of a fluid stream 46could provide, in a single image, enough information to determine ormodify drop delays.

The sensing element 48 may be any sensing element 48 capable ofconverting an image into a series of electrical or digital signals. Asone example, the sensing element 48 may be a charge coupled device(CCD). A charge coupled device may operate to produce a series of analogpulses based upon the light intensity received at each pixel of the CCD.These pulses may then be digitized to produce a digital image.Similarly, a complementary metal-oxide-semiconductor (CMOS) may be usedfor detecting pulses at individual pixels. Other sensors andconfigurations for detecting the light intensity of an image in highresolution may also be used, such as a photodiode array or a sensorarray.

Turning to FIG. 2, an imaging device 102 is illustrated which may beused for imaging a fluid stream 18 of a flow cytometer 10. Anillumination source, such as a strobe 80, laser, lamp, or LED strobe,may illuminate the fluid stream 18 periodically, or on demand, to ensurean image of the fluid stream 46 is produced. The image of the stream 46may be manipulated through an optical system 44 comprising a firstoptical element 52 and a second optical element 54 for producing amanipulated image of the fluid stream 50 incident upon the sensingelement 48. Each of the first optical element 52 and the second opticalelement 54 may be refractive optical elements, reflective opticalelements, or diffractive elements. FIG. 2 illustrates the first opticalelement 52 as a first cylindrical lens 60 for manipulating the image ofthe stream 46 in a first axis 62. Relative to the image of the fluidstream 46 depicted in FIG. 2, the first axis 62 may be referred to asthe X-axis. Throughout the following description the first axis 62 willcorrespond to the width 70 of the fluid stream 18, but the designationas the X-axis may be considered arbitrary. The manipulated image of thefluid stream 50 may retain its dimension in a second axis 66, such asthe Y-axis, which is orthogonal to the first axis 62, after passingthrough the first cylindrical lens 60. FIG. 2 illustrates the firstcylindrical lens in two different planes, and the different effect thefirst cylindrical lens 60 has on light in each plane is illustrated, asdescribed above. As one example, the first cylindrical lens 60 may be a25 mm cylindrical lens, such as one available from OptoSigma, partnumber 022-0160. The first cylindrical lens 60 may magnify the width 70of the image of the fluid stream 46 by between 3 times and 20 times, ormore.

The second optical element 54 may comprise a second cylindrical lens 64for manipulating the dimensions of the image of the fluid stream 46 in asecond axis 66. The second cylindrical lens 64 is illustrated in twodifferent planes as substantially aligned with first cylindrical lens 60and the combined effect is illustrated in the direction of themanipulated image of the fluid stream 50 in both planes. As anon-limiting example, the second cylindrical lens 64 may reduce thelength 68 of the image of the fluid stream 46 in the second axis 66,without further changing the width 70 of the image of the fluid stream46 in the first axis 62. As one example, the second cylindrical lens 64may be a 40 mm lens, such as one available from OptoSigma, part number022-0290. The second cylindrical lens 64 may magnify the stream length68 by magnitude of between 0.75 and 0.125. The second cylindrical lens64 may be seen as compressing the length 68 of the image of the fluidstream 46 by a factor between ¼ and 10.

The cylindrical lenses may cooperate to manipulate the aspect ratio ofthe image of the fluid stream 46 to a ratio of about 10 to 1. Variouscombinations of magnifications may be used to achieve this ratio, orother ratios. As one non-limiting example, the length 68 of the image ofthe fluid stream 46 may be magnified by a factor of 0.5 and the width 70of the image of the fluid stream 46 may be magnified by a factor of 5.Other configurations are contemplated herein, such those for providingaspect ratios of 2 to 1, 4 to 1, 10 to 1, 15 to 1, 20 to 1 or evenhigher. The optical elements may be placed in any order.

Referring now to FIG. 3, an imaging device 102 is illustrated havingoptics system 44′ with a first optical element 52′ in the form of afirst reflective element 72 and a second optical element 54′ in the formof a second reflective element 74. The reflective elements may compriseminors and particularly mirrors for manipulating the aspect ratio of theimages of the fluid stream 46. The first reflective element 72 maycomprise a mirror for expanding the width 70 of the image of the fluidstream 46 in a manner similar to that described with respect to FIG. 1.The second reflective element may comprise a minor for reducing thelength 68 of the image of the fluid stream 46 in a manner similar tothat described with respect to FIG. 1. It should be appreciated withrespect to the reflective and the refractive elements that the firstoptical element may be either for manipulating the length 70 of theimage of the fluid stream 46 or the width 68 of the image of the fluidstream 46. Further, embodiments contemplated herein include combinationsof refractive elements and reflective elements, such as, one or morecylindrical lenses for modifying one dimension of the image of the fluidstream and one or more mirrors for modifying another dimension of theimage of the fluid stream 46.

In still another embodiment, diffractive elements may be used formanipulating the aspect ratio of the image of the fluid stream 46. Itshould be understood that other optics for manipulating the aspect ratioof the image of the fluid stream 46 may be used in conjunction theoptics system 44 to deliver the image of the fluid stream 46 to theoptical system 44 or to the sensing element 48.

Combinations of optical elements may be used to modify the aspect ratiosof the manipulated image of the fluid stream 50 differently at differentpoints along the length 68 of the fluid stream 18. FIG. 4 illustrates amanipulated image of the fluid stream 50 produced by an optical system44 configured for continuous gradient. A first aspect ratio 108 at thetop of the manipulated image of the fluid stream 50, may remain 1 to 1,while the a second aspect ratio 110 near the break off point 30, may be5 to 1, 10 to 1, or another ratio. Resolving the width of the fluidstream 18 near a break off point well, may increase the accuracy ofdetecting the break off point 30 and changes in the break off point 30.A continuous gradient may be created with a combination of lenses andminors as well as with one or more gradient index GRIN lenses.

FIG. 5 illustrates a disjointed image, whereby a first portion of themanipulated image of the fluid stream 50 is transmitted at a firstaspect ratio 108, such as 1 to 1, and a second portion of themanipulated image of the fluid stream 50 is transmitted at a secondaspect ratio 110, such as 5 to 1 or 10 to 1. It should be appreciated;the disjointed image may be a compilation of multiple images havingdifferent aspect ratios, such as 3, 4, 5, 8, 10 or more images havingdifferent aspect ratios.

Each of the images created by the optics of FIG. 2 or FIG. 3 may betransmitted onto a single sensing element with or without magnification.Alternatively, the manipulated image of the fluid stream 50 may beprojected onto multiple sensing elements or a high resolution sensingelement described in more detail below.

Referring to FIG. 6, an imaging device 102 is illustrated with anoptical system 44″ which does not manipulate the aspect ratio of theimage of the fluid stream 46. Instead, the optic system 44″ may providefor a zoom factor of 10 for projecting an image of the fluid stream 46onto a high resolution sensing element 96. The high resolution sensingelement 96 may comprise a high resolution CCD, such as one havingbetween 0.3 megapixels and 20 megapixels or more. By way of non-limitingexamples the CCD may have 10 megapixels or 20 megapixels. The image ofthe fluid stream 46 may then be manipulated at the level of the highresolution sensing element 96 through a cropping function. The croppedhigh resolution image 104 may provide a manipulated aspect ratio similarto those manipulated with the optics systems 44 previously described.

Referring now to FIG. 7 an imaging device 102 is illustrated with asingle optical system 44′″ and two or more spatially separated sensingelements 100 a, 100 b. The single optics system 44′″ may manipulate theaspect ratio of the image of the fluid stream 46, or may maintain a 1 to1 aspect ratio with respect to the fluid steam length 68 and width 70.The single optics system 44′″ may also provide magnification, or maymerely focus the image of the fluid stream 46. FIG. 7 illustrates theimage of the fluid stream 46 with a manipulated aspect ratio, but theimages may also be magnified, or some combination thereof.

It may be of particular interest in a system having multiple sensingelements 100 to ensure the most important regions of interest fall onthe sensing elements 100 and not in any gap created by spacing. Forexample, it may be desirable to ensure the inspection zone 34 is wellcaptured on one sensing element and that the break off point 30 is wellcaptured on another sensing element. This may help to ensure the breakoff point 30 is accurately measured and the distance D between theinspection zone 34 and the break off point 30 is accurately determined.In one embodiment, the sensing element 100 corresponding to the breakoff point 30 may operate at an improved resolution compared to the othersensing elements. In another embodiment, the portion of the image withthe droplet break off 30 may be capture on a separate camera or sensingelement. Other optical configurations where portions of the image may besplit to multiple sensing elements that have an overlapping field ofview within the object plane may also be employed where, when processed,or recombined, there are no discontinuities in the image.

FIG. 8 depicts an embodiment of a method where a high resolution sensingelement 96 may be used in conjunction with high speed data acquisitionand processor in order to take multiple images of a forming droplet andto determine the precise time at which each droplet breaks way from thesteam, as shown in step 210. Such high speed computations may be made onthe central processing unit of a computer, on a single digital signalprocessor or on a field programmable gate array. Any of these processorsmay be programmed to modify the calculated drop delay based on digitalprocessing of one or more images.

The high resolution sensing element 96 may form a portion of a highspeed, high resolution camera which may be used in conjunction with aflow cytometer 10, like the one depicted in FIG. 1. The microprocessormay be located in the controller 38 or elsewhere, and may be incommunication with the high resolution sensing element 96 for extractingfeatures from the high resolution image and making determinations as tothe time at which the next droplet will break away from the fluidstream. High repetition rate or on-demand images may be captured andtransferred at a sufficiently high rate into memory or other suitablestorage mechanisms for real time processing, as illustrated in step 212.At step 214, data processing algorithms may be used to extract certainfeatures such as the thickness of the droplets or the width of the neckwhich may provide valuable information regarding the time at whichdroplet will break from the fluid stream. As a non-limiting example, acertain region of interest may be processed to track the position of thelast neck of last attached drop. Multiple images may be acquired andprocessed per droplet formation cycle so that the droplet formationprogress can be tracked and estimated, particularly the drop delay, asillustrated at step 216. Finally, with sufficient resolution, at step218 the processor may modify the drop delay on a droplet-by-dropletbasis as a function of processing the plurality of images to determinethe point at which an individual droplet is about to break free from thecontinuous fluid stream. These measurements may be particularly usefulwhen increased chaotic behavior is observed as particles are flowingwithin the fluid stream. It may not be necessary to observe the actualdroplet formation, or wait for actual droplet to form, since certainlatency exists within electronic circuitry. In fact, one may engage thecharge circuitry early to ensure that a suitable voltage level will beapplied to the continuous fluid stream prior to the droplet formationevent. The processor may then modify the existing predicted drop delay,or the calibrated or average drop delay, based upon this information. Apredictive model may also be generated from these observations.

Similarly, the processor may modify the drop delay on adroplet-by-droplet basis as a function of processing the plurality ofimages to determine the point at which the fluid stream between twoforming droplets becomes disconnected.

The high speed camera may take pictures as rapidly as between everymicrosecond and every 50 microseconds. In one embodiment, the high speedcamera can operate in two modes: a coarse mode and a fine mode. Thecoarse mode may apply when a droplet has just finished forming and thefine mode may apply when a droplet is approaching the break off point.The high speed camera may operate to take, or process, images morerapidly in fine mode than in coarse mode. The high speed camera mayoperate in phase or out of phase with the droplet formation signal orwith a particular phase offset.

The processor may include an image processing algorithm in the form ofwritten executable computer instructions for determining the time atwhich the next droplet will break off the fluid stream. The imageprocessing algorithm may be in the form of instructions stored on adigital signal processor, or a bitmap look up table on a fieldprogrammable gate array. The algorithm may define the periods for coarsemode and fine mode, whereby based upon the features extracted from theimages, the algorithm determines the rate at which images should becaptured. Images may be captured at two discrete rates or there may be acontinuous transfer between two or more rates. The algorithm mayselectively utilize individual images from a plurality of image capturedevents.

Features extracted from the images of the fluid stream 46 may include:edge detection, fluid stream features, droplet shape and position, neckgeometry and position, aspect ratio, contrast, statisticalcharacteristics such as means and standard deviation any parameter, andother features which may be extracted from the images.

As can be easily understood from the foregoing, the basic concepts ofthe present invention may be embodied in a variety of ways. Theinvention involves numerous and varied embodiments of flow cytometryacquisition and sort electronics and methods including, but not limitedto, the best mode of the invention.

As such, the particular embodiments or elements of the inventiondisclosed by the description or shown in the figures or tablesaccompanying this application are not intended to be limiting, butrather exemplary of the numerous and varied embodiments genericallyencompassed by the invention or equivalents encompassed with respect toany particular element thereof. In addition, the specific description ofa single embodiment or element of the invention may not explicitlydescribe all embodiments or elements possible; many alternatives areimplicitly disclosed by the description and figures.

It should be understood that each element of an apparatus or each stepof a method may be described by an apparatus term or method term. Suchterms can be substituted where desired to make explicit the implicitlybroad coverage to which this invention is entitled. As but one example,it should be understood that all steps of a method may be disclosed asan action, a means for taking that action, or as an element which causesthat action. Similarly, each element of an apparatus may be disclosed asthe physical element or the action which that physical elementfacilitates. As but one example, the disclosure of a “sensing element”should be understood to encompass disclosure of the act of“sensing”—whether explicitly discussed or not—and, conversely, werethere effectively disclosure of the act of “sensing”, such a disclosureshould be understood to encompass disclosure of a “sensing element” andeven a “means for sensing.” Such alternative terms for each element orstep are to be understood to be explicitly included in the description.

In addition, as to each term used it should be understood that unlessits utilization in this application is inconsistent with suchinterpretation, common dictionary definitions should be understood to beincluded in the description for each term as contained in the RandomHouse Webster's Unabridged Dictionary, second edition, each definitionhereby incorporated by reference.

Moreover, for the purposes of the present invention, the term “a” or“an” entity refers to one or more of that entity; for example, “acontainer” refers to one or more of the containers. As such, the terms“a” or “an”, “one or more” and “at least one” can be usedinterchangeably herein.

All numeric values herein are assumed to be modified by the term“about”, whether or not explicitly indicated. For the purposes of thepresent invention, ranges may be expressed as from “about” oneparticular value to “about” another particular value. When such a rangeis expressed, another embodiment includes from the one particular valueto the other particular value. The recitation of numerical ranges byendpoints includes all the numeric values subsumed within that range. Anumerical range of one to five includes for example the numeric values1, 1.5, 2, 2.75, 3, 3.80, 4, 5, and so forth. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint. When a value is expressed as an approximation by use of theantecedent “about,” it will be understood that the particular valueforms another embodiment.

Thus, the applicant(s) should be understood to claim at least: i) afluid stream imaging device for modifying the aspect ratio of an imageof a fluid, ii) an fluid stream imaging device with a high resolutionsensing element, iii) a flow cytometer with an optical system forimaging droplet formation and processing those images to determinedroplet break off times, iv) the related methods disclosed anddescribed, v) similar, equivalent, and even implicit variations of eachof these devices and methods, vi) those alternative embodiments whichaccomplish each of the functions shown, disclosed, or described, vii)those alternative designs and methods which accomplish each of thefunctions shown as are implicit to accomplish that which is disclosedand described, viii) each feature, component, and step shown as separateand independent inventions, ix) the applications enhanced by the varioussystems or components disclosed, x) the resulting products produced bysuch systems or components, xi) methods and apparatuses substantially asdescribed hereinbefore and with reference to any of the accompanyingexamples, and xii) the various combinations and permutations of each ofthe previous elements disclosed.

The background section of this patent application provides a statementof the field of endeavor to which the invention pertains. This sectionmay also incorporate or contain paraphrasing of certain United Statespatents, patent applications, publications, or subject matter of theclaimed invention useful in relating information, problems, or concernsabout the state of technology to which the invention is drawn toward. Itis not intended that any United States patent, patent application,publication, statement or other information cited or incorporated hereinbe interpreted, construed or deemed to be admitted as prior art withrespect to the invention.

The claims set forth in this specification, if any, are herebyincorporated by reference as part of this description of the invention,and the applicant expressly reserves the right to use all of or aportion of such incorporated content of such claims as additionaldescription to support any of or all of the claims or any element orcomponent thereof, and the applicant further expressly reserves theright to move any portion of or all of the incorporated content of suchclaims or any element or component thereof from the description into theclaims or vice versa as necessary to define the matter for whichprotection is sought by this application or by any subsequentapplication or continuation, division, or continuation-in-partapplication thereof, or to obtain any benefit of, reduction in feespursuant to, or to comply with the patent laws, rules, or regulations ofany country or treaty, and such content incorporated by reference shallsurvive during the entire pendency of this application including anysubsequent continuation, division, or continuation-in-part applicationthereof or any reissue or extension thereon.

The claims set forth in this specification, if any, are further intendedto describe the metes and bounds of a limited number of the preferredembodiments of the invention and are not to be construed as the broadestembodiment of the invention or a complete listing of embodiments of theinvention that may be claimed. The applicant does not waive any right todevelop further claims based upon the description set forth above as apart of any continuation, division, or continuation-in-part, or similarapplication.

We claim:
 1. A flow cytometer comprising: a. a fluid delivery systemhaving a nozzle for producing a fluid stream, the fluid delivery systemhaving an oscillator for perturbing the fluid stream into droplet at abreak off point downstream of the nozzle; b. an excitation energy sourcefor interrogating the fluid stream at an inspection zone; c. an opticalsystem for producing a image of the fluid stream, the image of the fluidstream including at least the inspection zone and the break off point;and d. a high resolution sensing element for sensing an image of thefluid stream.
 2. The flow cytometer of claim 1, wherein the highresolution sensing element has a resolution between 3 megapixels and 20megapixels.
 3. The flow cytometer of claim 1, wherein the aspect ratioof the high resolution image of the fluid stream sensed by the highresolution sensing element is manipulated.
 4. The flow cytometer ofclaim 1, wherein the high resolution sensing element comprises a singlehigh resolution CCD sensor for capturing the image of the fluid stream.5. The flow cytometer of claim 1, wherein the high resolution sensingelement comprises a single high resolution CMOS sensor for capturing theimage of the fluid stream.
 6. The flow cytometer of claim 1, wherein theimage of the fluid stream is cropped to modify the aspect ratio.
 7. Theflow cytometer of claim 1, wherein the high resolution sensing elementcomprises at least at least two equally spaced sensing elements each forsensing a portion of the image of the fluid stream produced by a commonoptical system.
 8. A flow cytometer comprising: a. a fluid deliverysystem having a nozzle for producing a fluid stream, the fluid deliverysystem having an oscillator for perturbing the fluid stream into dropletat a break off point downstream of the nozzle; b. an excitation energysource for interrogating the fluid stream at an inspection zone; c. adetector for detecting electromagnetic radiation from particles at theinspection zone and for determining particle characteristics; d. acontroller for charging the fluid stream based on the detected particlecharacteristics; e. an optical system for producing a image of the fluidstream, the image of the fluid stream including at least the break offpoint, wherein the optical system takes multiple images per droplet; f.a sensing element for sensing a plurality of images of the fluid stream;and g. a processor for processing the detected image of the fluidstream, wherein the processor is responsive to images of the fluidstream to determine the time at which a droplet breaks off the fluidstream.
 9. The flow cytometer of claim 8, wherein the optical system andthe sensing element comprises a high resolution camera.
 10. The flowcytometer of claim 8, wherein the sensing element includes multiplesensing elements.
 11. The flow cytometer of claim 8, wherein thecontroller applies a drop delay when charging the fluid stream forseparating particles and wherein the processor modifies the drop delayon a droplet-by-droplet basis as a function of processing the pluralityof images to determine the point at which an individual droplet breaksfree from the continuous fluid stream.
 12. The flow cytometer of claim8, wherein the controller applies a drop delay when charging the fluidstream for separating particles and wherein the processor modifies thedrop delay on a droplet-by-droplet basis as a function of processing theplurality of images to determine the point at which the fluid streambetween two forming droplet becomes disconnected.
 13. The flow cytometerof claim 8, wherein the detection of the plurality of images isseparated by one selected from the group consisting of: 1 microsecond, 5microseconds, 10 microseconds, 20 microseconds, and 50 microseconds. 14.The flow cytometer of claim 8, wherein the plurality of images isseparated in time depending on the state of the formation of the nextdroplet.
 15. The flow cytometer of claim 14, wherein the plurality ofimages is separated by less time near the break off of the next droplet.16. The flow cytometer of claim 8, wherein the time at which a dropletbreaks off the fluid stream as determined by the processor is furtherused by the processor to modify an average drop delay, a previouslycalibrated drop delay, or a previously assumed drop delay.
 17. The flowcytometer of claim 8, wherein the processor is configured with an imageprocessing algorithm to determine the time at which a droplet will breakoff the fluid stream.
 18. The flow cytometer of claim 8, wherein thealgorithm comprises a coarse determination and a fine determination. 19.The flow cytometer of claim 18, wherein images are captured at a firstrate to make a coarse determination and wherein images are captures at asecond faster rate to make a fine determination.
 20. The flow cytometerof claim 18, wherein the algorithm extracts one from selected from thefollowing features from the plurality of images to determine the timewhen a droplet will break off the fluid stream: edge detection, fluidstream features, droplet shape, aspect ratio, contrast, and combinationsthereof.